Abstract A central question in the field of Evo-Devo is how genes controlling embryonic development change during evolution. Many recent advances in Evo-Devo have identified genetic changes that are associated with the acquisition of, or changes in, external body features, such as alterations in pigmentation patterns or development of body armor. In contrast, our studies are novel in this field as they have revealed unexpected genetic variation underlying a highly conserved trait: the shared segmented body plan of insects. The genes controlling segmentation encode transcription factors that are required for embryonic development and viability. Thus, it has been surprising to find large differences in the presence, expression, or function of these genes in different insect taxa. The work proposed here is designed to understand the mechanistic changes underlying this observed genetic variation, in different insect lineages. To carry out functional studies, we have developed molecular genetic approaches in diverse insect species in our lab. The establishment of multiple non-model systems simultaneously within one lab has synergistic effects due to sharing of protocols and troubleshooting strategies, allowing us to more effectively develop new techniques in different species. With these tools in hand, we will examine the underlying bases of specific scenarios of regulatory gene variation: Aim 1. Re-wiring of gene regulatory interactions that permit loss of a conserved regulator in mosquitoes; Aim 2. Stability of target gene regulation despite switches in transcription factor partners: Ftz-F1 regulates target genes without Ftz in Tribolium. Aim 3. Regulation of segmentation by non-canonical PRGs in Hemiptera. These studies will contribute to our understanding of fundamental mechanisms regulating embryonic development and how these mechanisms have changed during the radiation of insects. This project will train postdoctoral fellows, one graduate student, and at least four undergraduate students in molecular biology, genetics and molecular evolution. Establishment of molecular techniques in non-model and emerging model insect species, including expression analysis, RNA interference, CRISPR, FAIRE-seq, and transgenesis, not only allows us to answer fundamental questions about embryonic development, but also provides molecular tools for translational studies of insects that pose a risk to human health.